A novel thermostable arylesterase from the archaeon Sulfolobus solfataricus P1: purification, characterization, and expression.
A novel thermostable arylesterase, a 35-kDa monomeric enzyme, was purified from the thermoacidophilic archaeon Sulfolobus solfataricus P1. The optimum temperature and pH were 94 degrees C and 7.0, respectively. The enzyme displayed remarkable thermostability: it retained 52% of its activity after 50 h of incubation at 90 degrees C. In addition, the purified enzyme showed high stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme has broad substrate specificity besides showing an arylesterase activity toward aromatic esters: it exhibits not only carboxylesterase activity toward tributyrin and p-nitrophenyl esters containing unsubstituted fatty acids from butyrate (C(4)) to palmitate (C(16)), but also paraoxonase activity toward organophosphates such as p-nitrophenylphosphate, paraoxon, and methylparaoxon. The k(cat)/K(m) ratios of the enzyme for phenyl acetate and paraoxon, the two most preferable substrates among all tested, were 30.6 and 119.4 s(-1) microM(-1), respectively. The arylesterase gene consists of 918 bp corresponding to 306 amino acid residues. The deduced amino acid sequence shares 34% identity with that of arylesterase from Acinetobacter sp. strain ADP1. Furthermore, we successfully expressed active recombinant S. solfataricus arylesterase in Escherichia coli. Together, our results show that the enzyme is a serine esterase belonging to the A-esterases and contains a catalytic triad composed of Ser156, Asp251, and His281 in the active site.
Park
Young-Jun
YJ
Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon 200-701, South Korea.
Yoon
Sung-Jin
SJ
Lee
Hee-Bong
HB
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D019641:DNA, Archaeal / Q000235:genetics; D004795:Enzyme Stability; D019766:Genes, Archaeal; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016415:Sequence Alignment; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D002265:Carboxylic Ester Hydrolases; C019734:arylesterase